Colony stimulating factor (CSF), as a glycoprotein acting on production, division, and activity of hematopoietic cell, is classified into granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), multi colony stimulating factor (multi-CSF), etc.
It is known that G-CSF increases releasing of matured neutrophils into peripheral blood by facilitating production and division of neutrophilic precursor at bone marrow; induces a phagocytic activity by activating matured neutrophils; represents an antibody dependent cell mediated cytotoxicity; generates superoxides; and increases reactivity against chemotactic factor.
Human granulocyte colony stimulating factor (hG-CSF) was firstly separated from bladder carcinoma cell line 5637 by Welt et al. in 1985, and recombinant hG-CSF (rhG-CSF) was produced by cloning of gene from cDNA library of a cancer cell by Nagata et al., Souza et al. in 1986. rhG-CSF currently used in clinical trials is filgrastim binding methionine at N-terminal of protein without glycosylation, produced in Escherichia coli, and lenograstim with glycosylation, produced in animal cell.
In case of using rhG-CSF clinically, rhG-CSF has to be administrated once or more a day for decreasing leukocyte because rhG-CSF has a short duration of pharmacological effect. Thus, many studies have been conducted to increase a half-life of circulation of rhG-CSF using water-soluble polymer, thereby developing the drug to have a long duration of activity and high stability, and decreasing the frequency of administration.
And, polyethylene glycol in the water-soluble polymer is strongly hydrophilic, and can increase solubility at the time of bonding with protein for treatment. Also, polyethylene glycol is effective for increasing the molecular amount of protein bonded thereto, with maintaining main biological functions such as enzyme activity and receptor binding. Thus, polyethylene glycol can decrease the filtration of kidney, and effectively protect protein from proteolytic enzyme to decompose the protein. Therefore, many studies have been conducted to find out modifying methods of protein by using polyethylene glycol because it has the advantages to prevent protein decomposition, increase the stability and circulation time of protein, and decrease immunogenicity.
Korean Patent No. 0508358 disclosed conjugates and a preparing method thereof, wherein the conjugates have biological activities and are in the form of binding a biocompatibility polymer to thiol of cysteine residue of G-CSF, in the molar ratio of the polymer:G-CSF to stoichiometrically 0.7˜1.3:1, preferably 1:1. But, since the cysteine residue of G-CSF which does not form disulfide bonds in G-CSF is main bonding part with G-CSF receptor, the conjugate using the cysteine residue has a drawback that its actual proliferation effect for neutrophil is little. And, because the conjugates immediately aggregate to bind a PEG to cysteine residues of G-CSF, it has a disadvantage that a small amount of SDS should be added to the conjugate to prevent the aggregation for safekeeping and the SDS was removed by ultrafiltration for administrating in vivo.
Korean Patent No. 0507796 disclosed PEG-homodimer conjugates binding a biological active polypeptide and a PEG, to increase the half-life in vivo of polypeptide. Particularly, it described that the homodimer binds an amino group of lycine residues of bioactive polypeptide of two molecules to a PEG to increase the residual time and sustain biological activities for a long time. But, the conjugates have less activity than mono-PEG conjugates because of conjugation of excessive PEGs and physicochemical and biological characteristics of the conjugates are not uniform because of non-specific conjugation.
And, the method of binding rhG-CSF to linear SCM-MPEG (Succinimidyl carboxymethyl ester of methoxy PEG) is known. The mono-PEG-G-CSF conjugates prepared by the method have 3 types of position isomers modified at N-terminal, lycine 35, and lycine 45 residues. Particularly, the conjugate modified at lycine 35 has a disadvantage leaving PEGs from the conjugate (Korean Patent No. 0248111, U.S. Pat. No. 5,824,784).
U.S. Pat. No. 5,951,974 disclosed conjugates of binding a linear SC-PEG (polyethylene glycol-N-succinimide carbonate) to gene recombination alpha interferon. The conjugates consist of covalent conjugates of urethane bond at ε-amino group of lycine residues of interferon, α-amino group of N-terminal cystaine residues, and an amino group of histidine residues. But, the conjugates have a disadvantage leaving PEGs from the conjugate because the urethane bond of conjugates are unstable.
Commonly used linear polyethylene glycol has a molecular weight of about 1,000˜25,000 daltons, but has a limitation in binding many linear high molecules to protein or peptide, with maintaining their activities, due to limited biological active regions of protein and peptide.
Korean Patent No. 0254097 disclosed conjugates binding a two-branched PEG of a lycine skeletal structure to gene recombination alpha interferon. The conjugates have a merit to prevent PEG from binding to multi parts of interferon, and have 2 times the molecular weight of PEG by that of the linear PEG because two linear PEGs bind to a single part of interferon. But, the two-branched PEG can be hydrolyzed to a single chain for safekeeping or for reacting under an alkali condition because the two-branched PEG having a lycine skeletal structure has two urethane bonds in the PEG.
Generally, it is known that biological activity, durability, etc. of conjugates of binding a PEG to gene recombination protein is dependent on size and part modified in the PEG. But, it has not been known yet whether a size of PEG in conjugates binding PEG and rhG-CSF affects biological activities of a protein. Therefore, there has been a need in the art to overcome the disadvantages of the known method through controlling a size of PEG and binding to various skeletal structures of PEG not to decrease bioactivities of G-CSF and to increase the stability of binding parts.